Efficacy in Galleria mellonella Larvae and Application Potential Assessment of a New Bacteriophage BUCT700 Extensively Lyse Stenotrophomonas maltophilia

ABSTRACT In recent years, Stenotrophomonas maltophilia (S. maltophilia) has become an important pathogen of clinically acquired infections accompanied by high pathogenicity and high mortality. Moreover, infections caused by multidrug-resistant S. maltophilia have emerged as a serious challenge in clinical practice. Bacteriophages are considered a promising alternative for the treatment of S. maltophilia infections due to their unique antibacterial mechanism and superior bactericidal ability compared with traditional antibiotic agents. Here, we reported a new phage BUCT700 that has a double-stranded DNA genome of 43,214 bp with 70% GC content. A total of 55 ORFs and no virulence or antimicrobial resistance genes were annotated in the genome of phage BUCT700. Phage BUCT700 has a broad host range (28/43) and can lyse multiple ST types of clinical S. maltophilia (21/33). Furthermore, bacteriophage BUCT700 used the Type IV fimbrial biogenesis protein PilX as an adsorption receptor. In the stability test, phage BUCT700 showed excellent thermal stability (4 to 60°C) and pH tolerance (pH = 4 to 12). Moreover, phage BUCT700 was able to maintain a high titer during long-term storage. The adsorption curve and one-step growth curve showed that phage BUCT700 could rapidly adsorb to the surface of S. maltophilia and produce a significant number of phage virions. In vivo, BUCT700 significantly increased the survival rate of S. maltophilia-infected Galleria mellonella (G. mellonella) larvae from 0% to 100% within 72 h, especially in the prophylactic model. In conclusion, these findings indicate that phage BUCT700 has promising potential for clinical application either as a prophylactic or therapeutic agent. IMPORTANCE The risk of Stenotrophomonas maltophilia infections mediated by the medical devices is exacerbated with an increase in the number of ICU patients during the Corona Virus Disease 2019 (COVID-19) epidemic. Complications caused by S. maltophilia infections could complicate the state of an illness, greatly extending the length of hospitalization and increasing the financial burden. Phage therapy might be a potential and promising alternative for clinical treatment of multidrug-resistant bacterial infections. Here, we investigated the protective effects of phage BUCT700 as prophylactic and therapeutic agents in Galleria mellonella models of infection, respectively. This study demonstrates that phage therapy can provide protection in targeting S. maltophilia-related infection, especially as prophylaxis.

provided new ideas for clinical application of phage therapy.
Title: "Clinical application potential" is over-interpreted. All the experiments are basic research but not pre-clinical studies.
The manuscript needs English editing. There have some grammatical errors in this manuscript that should be corrected carefully and thoroughly. 1)Italicized words Line 27, correct it as "in vivo". Line 78, correct it as "S.maltophilia". Note that Latin words should usually be printed in italics. Authors should check and correct all Latin words in the article. Line 215-216, correct it as "in vitro and in vivo". 2)Line 28, please replace "S.maltophilia infected" with "S.maltophilia-infected". 3)Line 32, please remove "the". 4)Line 66, please replace "for" with "of". 5)Line 174, please replace "DNA binding" with "DNA-binding". 6)Line 233, please replace "have" with "has". 7)The article "an" should be used in front of MOI instead of "a". 1.Line 29: I strongly suggest removing "G.mellonella larvae". 2.Line 79-81: The meaning of "phages" is not clear. I suggest replacing "phages" with " S.maltophilia phages". 3.Line 162: please remove "time". 4.Line 224-225: I suggest replacing "phage" with "phage BUCT700".
How to determine death of Galleria mellonella? How was the dose chosen? Provide more details and the references. If you have done different gradient dose examinations, please explain and provide the corresponding survival curve.
The the accession number should be listed in the "Data availability" Statistic statements are missing in the METHODS section. The figure legends are not very informative.

Reviewer #2 (Comments for the Author):
Infections caused by multidrug-resistant S. maltophilia have emerged as a serious challenge in clinical practice, Li et al. isolated a phage BUCT700 that can be effective for most clinical S. maltophilia isolates. The author well characterized this phage including one-step growth curve, replication kinetics, stability, host range, genome analysis and in vivo efficacy in a Galleria mellonella (G.mellonella) larvae model. This work demonstrated the potential of phage BUCT700 for future clinical utilization. However, the major concerns for this reviewer are the novelty and significance of this study to the readers of this journal. The data are not well presented in this manuscript.Figures are redundant and can be merged. The manuscript needs further improvement with the following suggestions: 1.Since the host bacteria S21 is not a standard strain but a clinical strain, the author should provide the characterization of this strain including the genome information, antibiogram and etc. 2.In the section of results "Genomic analysis and annotation of phage BUCT700", although authors try to describe the function of each annotated genes, this seems wordy, since these genes are general founded in other phages as well, the authors should focus on the specificity of the phage BUCT700. 3.Line 178, the author mixed the definition of endolysin and lysozyme, indeed the endolysin including the lysozyme. The author should check the annotation of the whole phage genome by interpro, HMMER or other tools for more accurate results. 4.Line 188, this sentence is not well written, change to "The lytic activity of phage BUCT700 with S. maltophilia S21" 5.The in vivo experimental result needs statistic analysis. 6.Figure 7 B and C is not well drawn, control group and phage only group is not separated in the figure. Please make a clearer marker. 7.Since the author found the phage BUCT700 is a wide host-range phage, it's a pity that the author did not investigate the receptor binding site for this phage, I suggest the author studying the receptor binding site for this phage. 8.All the sequences should be deposited in a public database with accession no.
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Incl.: Point-by-Point Responses to the Reviewer's Comments
Reviewer #1 (Comments for the Author): The authors provide a bacteriophage named BUCT700 which can lyse multiple clinical ST species of Stenotrophomonas maltophilia, which is a clinical challenge.
The safety and therapy potential of phage BUCT700 for clinical applications were evaluated by genomic level analysis and stability experiments. The authors used a Galleria mellonella model to examine the efficacy and safety of treatment with phage BUCT700 in vivo. Interestingly, phage BUCT700 was able to provide effective protection to Galleria mellonella. This is a rigorous and interesting work. The authors evaluated the potential for clinical application of phage BUCT700 and proposed various ways for clinical use of phage BUCT700 in the discussion section, which provided new ideas for clinical application of phage therapy.
Response: Thanks for your approbation of this manuscript, and we appreciate your constructive suggestions. We have addressed the issues you pointed out to improve the quality of our manuscript.
Title: "Clinical application potential" is over-interpreted. All the experiments are basic research but not pre-clinical studies.
Response: Thank you for your kind reminder. We have removed "Clinical" from the title and changed the title to "Efficacy in Galleria mellonella larvae and application potential assessment of a new bacteriophage BUCT700 extensively lyse Stenotrophomonas maltophilia".
The manuscript needs English editing. There have some grammatical errors in this manuscript that should be corrected carefully and thoroughly.
Response: Thank you for your constructive suggestions regarding our manuscript, we carefully checked the whole manuscript for any typos and polished all sections in current manuscript.

infection. In this study, we investigated for the first time the effect of phages as prophylactic agents in S.maltophilia-related infection models and found that phages as prophylactic agents provided better protection against S.maltophilia
infections compared with the treatment group ( Figure 8C and 8D). The results of evaluating phage BUCT700 as prophylactic agents showed that the survival rate of G.mellonella larvae was 100% at an MOI of 100, 96.15% at an MOI of 10, 93.33% at an MOI of 1, and 81.25% at an MOI of 0.1, respectively ( Figure 8D).
In summary, we believe that phage BUCT700 could be considered as a prophylactic agent to be added to the treatment regimen. Figure 8C. Survival rate on therapeutic treatment. Figure 8D. Survival rate on prophylactic treatment.  Response: Thank you for your kind reminder, we have checked the annotation of the whole phage genome by interpro (https://www.ebi.ac.uk/interpro/). We have changed the ORF54 to SAR endolysin (shown in Figure 5). 7.Since the author found the phage BUCT700 is a wide host-range phage, it's a pity that the author did not investigate the receptor binding site for this phage, I suggest the author studying the receptor binding site for this phage.